Interpretive Summary: Coliforms are a group of bacteria, which include Escherichia coli, Salmonella, and Shigella species – sewage-associated bacteria often found in foods, water, and environmental samples. In contrast, Vibrionaceae are a family of bacteria that are naturally present in the marine environment. Some species of Vibrionaceae cause illness and death among shellfish consumers. Rapid, simple, and cost-effective methods are needed to detect these organisms. This paper describes the development of three enzyme-based tests which determine total coliform levels, E. coli levels, and total Vibrionaceae levels in seawater and shellfish. In addition, a combined test method for total coliforms and total Vibrionaceae is described. These techniques involve the overlay of bacterial colonies growing on culture dishes with a membrane that detects two different enzymes produced by the bacteria. The presence of the enzyme causes fluorescent blue spots for the coliforms, including E. coli, and fluorescent green spots for the Vibrionaceae on the membranes within 10 to 30 min. These methods are practical for use in routine screening of foods, water, and environmental samples, since they are rapid, simple, and inexpensive. Membrane overlays could enhance food safety by allowing increased numbers of samples to be tested at a reduced cost or by providing a simple alternative to procedures that are too rigorous for routine use.

Technical Abstract:
Three assays were developed to enumerate total coliforms, Escherichia coli, and total Vibrionaceae in shellfish and other foods and in seawater and other environmental samples. Assays involve membrane overlays of overnight colonies on non-selective agar plates to detect ß-glucuronidase and lysyl aminopeptidase activities for coliforms and Vibrionaceae, respectively. Membranes containing the substrates 4-methylumbeferyl-ß-D-glucuronide produced a bright blue fluorescence when overlaid onto E. coli, while L-lysyl-7-amino-4-trifluoromethylcoumarin produced white or green fluorescent foci when overlaid onto Vibrionaceae family members, depending on the type of membrane used. A multiplex assay was also developed for simultaneously enumerating total coliforms and total Vibrionaceae in oysters and seawater. Overall, 65% of overlaid E. coli (non-O157:H7) were MUG-positive, compared with 62% as determined by the most-probable-number-MUG assay. The overlays are rapid (10 min), simple, and cost effective for quantification of total and fecal coliforms and Vibrionaceae. This research provides practical alternatives for monitoring bacterial indicators and potential pathogens in complex samples, including molluscan shellfish.